Several publications are referenced in this application by numerals in parenthesis in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Preconditioning (PC) with brief ischemia before a sustained period of ischemia has been shown to reduce infarct size in isolated perfused heart. This preconditioning phenomenon has been observed in perfused hearts from a number of mammalian species including dog, guinea pig, pig, rabbit, and rat (1-6, 10-13). Indirect evidence for protective preconditioning also exists in humans (5, 6, 14). Adenosine is released in large amounts during myocardial ischemia and has been demonstrated to play a major role in mediating preconditioning and other cardioprotective effects in most animal species including humans (1-14). Previous studies (15-17) of adult human and rabbit ventricular myocytes and cultured neonatal rat cardiac myocytes provided important insight by indicating that the cardioprotective mechanism of preconditioning is exerted, at least in part, at the level of and by the cardiac myocytes in the intact heart. Although a non-A.sub.1 receptor, possibly the A.sub.3 subtype, may be involved in mediating preconditioning, very little is known regarding the role of different adenosine receptor subtypes in mediating or modulating preconditioning of the cardiac myocytes.
Ventricular myocytes cultured from chick embryos retain many of the properties of the intact heart and have served as a useful model for a variety of experimental paradigms (18-24). Previous studies have demonstrated that activation of adenosine receptors in these cultured heart cells produces physiologic effects similar to those elicited by adenosine in the adult mammalian heart (25-28). The cultured ventricular myocytes contain predominantly (&gt;90%) myocytes (21) and are largely devoid of neuronal, blood or vascular cells, thus the confounding influence of changes in blood flow is avoided (18-28). This chick ventricular cell culture provides a model system to investigate the role of adenosine receptor subtypes in the preconditioning process and to study the mechanism(s) by which preconditioning of the ventricular myocytes can be modulated. To simulate preconditioning, ventricular myocytes were exposed to five minutes of hypoxia (O.sub.2 &lt;1%), reoxygenated in the presence of normal % O.sub.2 (room air) for ten minutes, and then incubated in the presence of continuous hypoxia for ninety minutes (O.sub.2 &lt;1%). The development of a ventricular myocyte model for preconditioning and the use of a protocol identical to that employed in preconditioning of the isolated perfused heart facilitates cellular characterization of this phenomenon and enables quantitative determination of the extent of cardioprotection by preconditioning.
Use of this model has facilitated the identification of compounds that enhance the protective effects of preconditioning and that increase myocardial resistance to ischemia.